Apolipophorin 1 1 1 in locusts : purification and character izat ion

Three molecular species of apolipophorin I11 were purified from adult locust hemolymph by gel filtration and ionexchange chromatography, and named apo-111-a, apo-111-b, and apo-111-c, respectively. They were indistinguishable by SDSpolyacrylamide gel electrophoresis, immunodiffusion, and in amino acid composition; however, they had different isoelectric points (5.43 for a, 5.11 forb, and 4.98 for c) and, therefore, could be separated by nativeor urea-gel electrophoresis. All three apo-111s were glycoproteins and contained fucose, mannose, and glucosamine. The total sugar content amounted to about 11% for each of the three apo-111s. The molecular weight of apo-I11 determined by SDS-polyacrylamide gel electrophoresis was approximately 20,000, almost equivalent to the native molecular weight (approximately 19,000) estimated by the sedimentationequilibrium method.mThis indicated that the locust apo-I11 exists in hemolymph as a monomeric form. It was demonstrated that a total 9 moles of apo-I11 (2 moles apo-111-a, 6 moles apo111-b, and 1 mole apo-111-c) associate with each mole of lipophorin in response to the action of locust adipokinetic hormone. -Chino, H., and M. Yazawa. Apolipophorin I11 in locusts: purification and characterization. J. Lipid h. 1986. 27: 377-385. Supplementary key words diacylglycerol glycoprotein lipophorin insect adipokinetic hormone Lipophorin is the major lipoprotein in the hemolymph of most insects (l), and serves as a reusable shuttle to transport various lipids including diacylglycerol, hydrocarbons, cholesterol, and carotenoids between tissues (2-5). Lipophorin generally comprises two apoproteins, a heavy chain (apo-I) with a molecular weight of 250,000 and a light chain (apo-11) with a molecular weight of 80,00085,000 (2, 6, 7). Insect adipokinetic hormone (AKH), first discovered in locusts by Mayer and Candy (8), is released from the corpora cardiaca during flight and stimulates the loading by lipophorin of diacylglycerol from the fat body, resulting in the formation of larger lipophorin particles with increased diacylglycerol content (9-11). Injection of AKH into adult locusts causes the association of a low molecular weight, non-lipid-containing protein with lipophorin (12-15). Similar observations have been extended to the adult tobacco hornworm, Manduca sexta, by Shapiro and Law (16) who proposed the term apolipophorin I11 (apo111) for the low molecular weight protein that becomes associated with lipophorin in response to AKH injection. The purification of apo-I11 from the hemolymph of M. sexta has been recently achieved in their laboratory (17). Our previous report (18) extended these observations to the locust, in which we demonstrated that the injection of AKH promotes the association of free apo-I11 in hemolymph with lipophorin and the loading of diacylglycerol from the fat body by lipophorin to produce larger, lower density “lipophorin particles. The structural changes of lipophorin in response to AKH are completely reversible, so that within 24 hr of AKH injection, the apo-I11 dissociates from lipophorin and the size and density of lipophorin return to the original values observed in resting locusts The effect of AKH in locust has been demonstrated in vitro using partially purified lipophorin and crude Cpprotein (equivalent to apo-111) by Van Heusden, Van Der Horst, and Beenakkers (19). However, the mechanism by which the above structural changes of lipophorin are induced under the action of AKH still remains unresolved. The resolution of this mechanism requires the availability of purified apo-111. The present study describes the purification and characterization of locust apo-111. Evidence is also presented to indicate that locust apo-I11 largely differs from M. sexta apo-I11 in physico-chemical properties. (18). MATERIALS AND METHODS Animals and collection of hemolymph Adult locusts, Locurta mipatoria, (3-5 weeks after the final molt) were taken from colonies maintained in this laboratory. Hemolymph was collected from resting and Abbreviations: apo-111, apolipophorin 111; AKH, adipokinetic hormone; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Journal of Lipid Research Volume 27, 1986 377 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom AKH-injected locusts (90 min after injection) by the “flushing out” method (20).